This study examined the emergence and magnitude of resistance to the small molecule ML336 in Venezuelan equine encephalitis virus (VEEV). Next-generation sequencing (NGS) was used to evaluate population-level resistance profiles as VEEV was repeatedly passaged in the presence of ML336. SNPs and consensus sequences that were generated from NGS data were used to compare the effects of differing microenvironments on the evolutionary trajectory of the emergence of ML336 resistance.